Signal transduction by G-protein coupled receptors is being studied using leukocyte chemotaxis as a model. Stimulation of cells with chemoattractants results in increased superoxide production, changes in the organization of cytoskeletal proteins and changes in specific gene expression. Many proteins involved in these signaling pathways are modified by lipids and by carboxyl methylation. These posttranslational modifications provide novel targets for pharmacologic manipulation of signaling pathways controling cell replication, motility, neurotransmitter release, ion channels, and cell-cell interactions. A. Carboxyl methylation and methyl group turnover of cdc42 and rhoA in RAW264 macrophage cells. We previously demonstrated a GTP-dependent methylation of low-molecular weight GTP-binding proteins, in a reaction catalyzed by a membrane bound prenylcysteine methyltransferase. Methyl group turnover in intact cells was determined by labeling the cells with [3H-methyl]-methionine and immune precipitation of cdc42 and rhoA with specific antibodies. Both cdc42 and rhoA were greater than 90% methylated. Stimulation with serum, C5a, lysophosphatidic acid, and bradykinin did not significantly increase the methyl group turnover in these two proteins, indicating that methyl group turnover is not required for activation of these signaling pathways. B. Palmitoylation of Gs protein alpha subunit. The alpha subunit of Gs is modified by palmitoylation. An HPLC method was developed to resolve the modified and unmodified forms of Gs, in order to directly determine the fraction of the protein modified by the lipid. In S49 cells, most of the Gs alpha subunit is palmitoylated, and isoproterenol stimulated incorporation of radioactive palmitate into the Gs alpha subunit. However, the overall level of lipid modification remained constant indicating that the thioesterase is the rate-limiting step in palmitate turnover on Gs.